Developmental regulation of human-like globin gene expression is controlled by several factors, primarily the trans-acting transcriptional milieu and cis-acting DNA elements. Identifying these DNA sequences and understanding the cis regulatory mechanisms underlying globin gene switching are essential to the development of therapeutic strategies for treatment of hemoglobinopathies such as sickle cell disease and B-thalassemias. The parent grant to this project, R01 HL67336, "Locus-linked Regulatory Motifs of Globin Gene Switching," has been investigating the function of these DNA regulatory regions using transgenic mice or routine cell lines. However, studies of globin gene expression in human cell lines may provide novel insights into globin gene regulation not revealed in mouse systems. Thus, the goal of this supplement application is to implement the use of human embryonic stem cells (hESCs) for broad-based studies of B-like globin gene switching, with an initial emphasis on examining the role of the human B-globin LCR at its endogenous genomic location. Specific Aim 1 will establish hESC line H1 culture conditions and define the parameters necessary to derive erythroid cells from this line. In Specific Aim 2, a hESC line H1 genomic library will be constructed for use in current and future gene targeting applications. The goal of Specific Aim 3 will be to produce mutations within the LCR to test its function within the native human globin locus. This will be accomplished by deleting the LCR or replacing it with the CMV enhancer or the B-globin HS -40 control region. These modified ES cells will be used to test whether locus chromatin is open or closed in the absence of the B-globin LCR. Replacement of the LCR with alternate enhancers will test whether the human B-globin LCR functions exclusively as an enhancer or has additional roles in the temporal regulation of downstream genes. Specific Aim 4 will analyze the expression of the murine B-like globin genes in human cells to better understand the evolutionary relationships and conservation of gene regulation and expression patterns between these two species. [unreadable] [unreadable]